Potential for Using Histidine Tags in Purification of Proteins at Large Scale

• Published in: Chemical engineering & technology 2005-11, Vol.28 (11), p.1306-1314
• Main Authors: Gaberc-Porekar, V., Menart, V.
• Format: Article
• Language: English
• Subjects: Chromatography; Proteins; Purification
• ISSN: 0930-7516; 1521-4125
• DOI: 10.1002/ceat.200500167

Attachment of oligo‐histidine tag (His‐tag) to the protein N‐ or C‐terminus is a good example of early and successful protein engineering to design a unique and generalized purification scheme for virtually any protein. Thus relatively strong and specific binding of His‐tagged protein is achieved on an Immobilized Metal‐Ion Affinity Chromatography (IMAC) matrix. Most popular hexa‐histidine tag and recently also deca‐histidine tag are used in combination with three chelating molecules: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), and carboxymethyl aspartic acid (CM‐Asp), covalently attached to the chromatographic matrix. The following combinations with divalent metal ions are preferentially used: (Cu, Zn, Ni, Co)‐IDA, Ni‐NTA, and Co‐CM‐Asp. At large scale, regarding cost and product purity, a decisive step is precise and efficient cleavage of His‐tag by the cleavage enzyme. Two‐step IMAC followed by a polishing step appears to be a minimum but still realistic as an approach to generic technology also for more demanding products. Possible drawbacks in using His‐tags and IMAC, such as leaching of metal ions, inefficient cleavage, and batch‐to‐batch reproducibility must be carefully evaluated before transferred to large scale. Although a great majority of reports refer to small laboratory scale isolations for research purposes it appears there is much higher potential for more extensive use of His‐tags and IMAC at large scale than currently documented. By genetic manipulations it is easy to fuse an oligo‐histidine tag to the N‐ or C‐terminus of any protein to be produced in recombinant microorganisms at laboratory or large scale. The tag should simplify purification and reduce the number of chromatographic steps. Consequently, this should significantly increase the final yield and substantially reduce the investment and production costs. The review shows that there is a much higher potential for more extensive use of His‐tags and Immobilized Metal‐Ion Affinity Chromatography (IMAC) at large scale than currently documented.